The present invention relates to a new class of aminoglycosides and to uses thereof in the treatment of genetic disorders.
Many human genetic disorders result from nonsense mutations, where one of the three stop codons (UAA, UAG or UGA) replaces an amino acid-coding codon, leading to premature termination of the translation and eventually to truncated inactive proteins. Currently, hundreds of such nonsense mutations are known, and several were shown to account for certain cases of fatal diseases, including cystic fibrosis (CF), Duchenne muscular dystrophy (DMD), ataxia-telangiectasia, Hurler syndrome, hemophilia A, hemophilia B, Tay-Sachs, and more [1, 2]. For many of those diseases there is presently no effective treatment, and although gene therapy seems like a potential possible solution for genetic disorders, there are still many critical difficulties to be solved before this technique could be used in humans.
During the last several years, it has been shown that aminoglycosides could have therapeutic value in the treatment of several genetic diseases because of their ability to induce ribosomes to read-through stop codon mutations, generating full-length proteins from part of the mRNA molecules [3-6].
Aminoglycosides are highly potent, broad-spectrum antibiotics commonly used for the treatment of life-threatening infections [7, 8]. The 2-deoxystreptamine (2-DOS) aminoglycosides antibiotics, shown in background art FIG. 1 [9], selectively target the prokaryotic ribosome, and, by binding to the decoding A-site of the 16S ribosomal RNA, lead to protein translation inhibition and interference with the translational fidelity [7, 10-12]. One of the most studied aminoglycosides is paromomycin (its sulfate salt known under its brand name Humatin), which is an antimicrobial drug used against intestinal amebiasis. It was approved by the Drug Controller General of India as an agent against visceral leishmaniasis (kala azar) in India, and was granted “orphan drug” status in 2005 in the US. Paromomycin is known to inhibit protein synthesis by binding to the ribosomal RNA of the 16S subunit.
Several achievements in bacterial ribosome structure determination [13-17], along with crystal and NMR structures of bacterial A-site oligonucleotide models [18-22], have provided useful information for understanding the decoding mechanism in prokaryote cells and understanding how aminoglycosides exert their deleterious misreading of the genetic code. During decoding, a critical step in aminoacyl-tRNA selection is based on the formation of a mini-helix between the codon of the mRNA and the anti-codon of the cognate aminoacyl-tRNA. In this process, the conformation of the A-site is changed from an ‘off’ state, where the two conserved adenines A1492 and A1493 are folded back within the helix, to an ‘on’ state, where A1492 and A1493 are flipped out from the A-site and interact with the cognate codon-anticodon mini-helix [11, 15]. This conformational change is a molecular switch that decides on the continuation of translation in an irreversible way. The binding of aminoglycosides such as paromomycin and gentamicin to the bacterial A-site stabilizes the ‘on’ conformation even in the absence of cognate tRNA-mRNA complex. Thus, the affinity of the A-site for a non-cognate mRNA-tRNA complex is increased upon aminoglycosides binding, preventing the ribosome from efficiently discriminating between non-cognate and cognate complexes.
The termination of protein synthesis is signaled by the presence of a stop codon in the mRNA, and is mediated by release factor proteins. The efficiency of translation termination is usually very high, and in intact cells the misincorporation of an amino acid at a stop codon (suppression) normally occurs at a low frequency of around 10−4. The enhancement of termination suppression by aminoglycosides in eukaryotes is thought to occur in a similar mechanism to the aminoglycosides' activity in interfering with translational fidelity during protein synthesis, namely the binding of certain aminoglycosides to the ribosomal A-site probably induce conformational changes that stabilize near-cognate mRNA-tRNA complexes, instead of inserting the release factor. Aminoglycosides suppress the various stop codons with notably different efficiencies (UGA>UAG>UAA), and the suppression effectiveness is further dependent upon the identity of the fourth nucleotide immediately downstream from the stop codon (C>U>A≥G) as well as the local sequence context around the stop codon [6, 23].
The fact that aminoglycosides could suppress premature nonsense mutations in mammalian cells was first demonstrated by Burke and Mogg in 1985, who also noted the therapeutic potential of these drugs in the treatment of genetic disorders [3].
The first genetic disease examined was cystic fibrosis (CF), the most prevalent autosomal recessive disorder in the Caucasian population, affecting 1 in 2,500 newborns. CF is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Currently, more than 1,000 different CF-causing mutations in the CFTR gene were identified, and 5-10% of the mutations are premature stop codons. In Ashkenazi Jews, the W1282X mutation and other nonsense mutations account for 64% of all CFTR mutant alleles [5].
The first experiments of aminoglycoside-mediated suppression of CFTR stop mutations demonstrated that premature stop mutations found in the CFTR gene could be suppressed by G-418 and gentamicin (see, background art FIG. 1), as measured by the appearance of full-length, functional CFTR in bronchial epithelial cell lines [24, 25]. Suppression experiments of intestinal tissues from CFTR−/− transgenic mice mutants carrying a human CFTR-G542X transgene showed that treatment with gentamicin, and to lesser extent tobramycin, have resulted in the appearance of human CFTR protein at the glands of treated mice [26]. Most importantly, clinical studies using double-blind, placebo-controlled, crossover trails have shown that gentamicin can suppress stop mutations in affected patients, and that gentamicin treatment improved transmembrane conductance across the nasal mucosa in a group of 19 patients carrying CFTR stop mutations [27]. Other genetic disorders for which the therapeutic potential of aminoglycosides was tested in in-vitro systems, cultured cell lines, or animal models include DMD [28], Hurler syndrome [29], nephrogenic diabetes insipidus [30], nephropathic cystinosis [31], retinitis pigmentosa [32], and ataxia-telangiectasia [33].
However, one of the major limitations in using aminoglycosides as pharmaceuticals is their high toxicity towards mammals, typically expressed in kidney (nephrotoxicity) and ear-associated (ototoxicity) illnesses. The origin of this toxicity is assumed to result from a combination of different factors and mechanisms such as interactions with phospholipids, inhibition of phospholipases and the formation of free radicals [34, 35]. Although considered selective to bacterial ribosomes, most aminoglycosides bind also to the eukaryotic A-site but with lower affinities than to the bacterial A-site [36]. The inhibition of translation in mammalian cells is also one of the possible causes for the high toxicity of these agents. Another factor adding to their cytotoxicity is their binding to the mitochondrial 12S rRNA A-site, whose sequence is very close to the bacterial A-site [37].
Many studies have been attempted to understand and offer ways to alleviate the toxicity associated with aminoglycosides [38], including the use of antioxidants to reduce free radical levels [39, 40], as well as the use of poly-L-aspartate [41, 42] and daptomycin [43, 44] to reduce the ability of aminoglycosides to interact with phospholipids. The role of megalin (a multiligand endocytic receptor which is especially abundant in the kidney proximal tubules and the inner ear) in the uptake of aminoglycosides has recently been demonstrated [35]. The administration of agonists that compete for aminoglycoside binding to megalin also resulted in a reduction in aminoglycoside uptake and toxicity [45]. In addition, altering the administration schedule and/or the manner in which aminoglycosides are administered has been investigated as means to reduce toxicity [46, 47].
Despite extensive efforts to reduce aminoglycoside toxicity, few results have matured into standard clinical practices and procedures for the administration of aminoglycosides to suppress stop mutations, other than changes in the administration schedule. For example, the use of sub-toxic doses of gentamicin in the clinical trails probably caused the reduced read-through efficiency obtained in the in-vivo experiments compared to the in-vitro systems [48]. The aminoglycoside Geneticin® (G-418 sulfate, see, background art FIG. 1) showed the best termination suppression activity in in-vitro translation-transcription systems [6], however, its use as a therapeutic agent is not possible since it is lethal even at very low concentrations. For example, the LD50 of G-418 against human fibroblast cells is 0.04 mg/ml, compared to 2.5-5.0 mg/ml for gentamicin, neomycin and kanamycin [49].
The increased sensitivity of eukaryotic ribosomes to some aminoglycoside drugs, such as G-418 and gentamicin, is intriguing but up to date could not be rationally explained because of the lack of sufficient structural data on their interaction with eukaryotic ribosomes. Since G-418 is extremely toxic even at very low concentrations, presently gentamicin is the only aminoglycoside tested in various animal models and clinical trials. Although some studies have shown that due to their relatively lower toxicity in cultured cells, amikacin [50] and paromomycin [51] can represent alternatives to gentamicin for stop mutation suppression therapy, no clinical trials with these aminoglycosides have been reported yet.
To date, nearly all suppression experiments have been performed with clinical, commercially available aminoglycosides [6], and no efforts have been made to optimize their activity as stop codon read-through inducers. Currently, only a limited number of aminoglycosides, including gentamicin, amikacin, and tobramycin, are in clinical use as antibiotics for internal administration in humans. Among these, tobramycin do not have stop mutations suppression activity, and gentamicin is the only aminoglycoside tested for stop mutations suppression activity in animal models and clinical trials. Recently, a set of neamine derivatives were shown to promote read-through of the SMN protein in fibroblasts derived from spinal muscular atrophy (SPA) patients; however, these compounds were originally designed as antibiotics and no conclusions were derived for further improvement of the read-through activity of these derivatives [52].
U.S. patent application Ser. No. 11/073,649, by the present assignee, which is incorporated by reference as if fully set forth herein, teaches a family of aminoglycosides, which have common structural backbone features which enables these aminoglycosides to be highly potent and effective antibiotics, while reducing or blocking antibiotic resistance thereto. The aminoglycoside derivatives taught in U.S. patent application Ser. No. 11/073,649, are presented as effective antibiotics against bacterial infections such as anthrax, and also as therapeutic agents for the treatment of genetic disorder, such as cystic fibrosis.
More specifically, the compounds taught in U.S. patent application Ser. No. 11/073,649 were designed based upon known aminoglycosides antibiotics which exert their antibacterial activity by selectively recognizing and binding to the decoding A site on the 16S subunit of the bacterial rRNA. Thus, these compounds are semi-synthetic analogs of currently available aminoglycosides, in which a pre-determined position of the aminoglycoside has been modified so as to enhance the recognition of the phosphodiester bond of rRNA and in parallel the Asp/Glu and Asn/Gln clusters in the active site of the lethal factor (LF) and thereby exhibit enhanced anti-bacterial performance. These modifications further provide the compounds with resistance to enzymatic catalysis and thus improve their bioavailability and hence anti-bacterial performance. Furthermore, the steric hindrance introduced into the designed structures via the chemical modification of the aminoglycoside, renders these compounds inferior substrates for the most widely represented resistance-causing enzyme, APH(3′)-IIIa, thus preventing the development of resistance thereto.
The design and bifunctional activity of these structures is also described by Mariana Hainrichson et al, in Bioorganic and Medicinal Chemistry 13 (2005) 5797-5807.
The compounds taught in the compounds taught in U.S. patent application Ser. No. 11/073,649 were further found to block a premature stop codon and hence effective in treating genetic disorders. However, as detailed hereinbelow, the enhanced antibacterial activity of these compounds may be undesirable when used to treat genetic disorders. Other modified aminoglycosides and structurally related antibiotics have been proposed and prepared [53-61] yet the stop-codon read-through therapeutic activity thereof was neither described nor suggested or tested.
The desired characteristics of an effective read-through drug would be oral administration and little or no effect on bacteria. Antimicrobial activity of read-through drug is undesirable as any unnecessary use of antibiotics, particularly with respect to the gastrointestinal (GI) biota, due to the adverse effects caused by upsetting the GI biota equilibrium and the emergence of resistance. In this respect, in addition to the abovementioned limitations, the majority of clinical aminoglycosides are greatly selective against bacterial ribosomes, and do not exert a significant effect on cytoplasmic ribosomes of human cells.
In an effort to circumvent the abovementioned limitations, the biopharmaceutical company PTC Therapeutics (NY, USA) is trying currently to discover new stop mutations suppression drugs by screening large chemical libraries for nonsense read-through activity. Using this approach, a new non-aminoglycoside compound, PTC124, was discovered [62].

The facts that PTC124 is reported to have no antibacterial activity and no reported toxicity, suggest that its mechanism of action on the ribosome is different than that of the aminoglycosides. The FDA has granted fast track and orphan drug designations to PTC124 for the treatment of both CF and DMD caused by nonsense mutations, and the preliminary results of phase II clinical trails in CF and DMD patients seem promising [63-67].